Apoptosis or "Programmed Cell Death"
Control of cell numbers in mammals is believed to be determined, in part, by a balance between cell proliferation and cell death. One form of cell death, sometimes referred to as necrotic cell death, is typically characterized as a pathologic form of cell death resulting from some trauma or cellular injury. In contrast, another "physiologic" form of cell death usually proceeds in an orderly or controlled manner. This orderly or controlled form of cell death is often referred to as "apoptosis" (see, e.g., Barr, et al., Bio/Technology, 12:487-493 (1994); Steller, et al., Science, 267:1445-1449 (1995)).
Apoptotic cell death naturally occurs in many physiological processes, including embryonic development and clonal selection in the immune system (Itoh, et al., Cell, 66:233-243 (1991)). Decreased levels of apoptotic cell death have been associated with a variety of pathological conditions, including cancer, lupus, and herpes virus infection (Thompson, Science, 267:1456-1462 (1995)). Increased levels of apoptotic cell death may be associated with a variety of other pathological conditions, including AIDS, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, retinitis pigmentosa, cerebella degeneration, aplastic anemia, myocardial infarction, stroke, reperfusion injury, and toxin-induced liver disease (see, Thompson, Supra).
Apoptotic cell death is typically accompanied by one or more characteristic morphological and biochemical changes in cells, such as condensation of cytoplasm, loss of plasma membrane microvilli, segmentation of the nucleus, degradation of chromosomal DNA or loss of mitochondrial function. A recognized biochemical marker of apoptosis is the cleavage of chromatin into nucleosomal fragments.
A variety of extrinsic and intrinsic signals are believed to trigger or induce such morphological and biochemical cellular changes (Raff, Nature, 356:397-400 (1992); Steller, Supra; Sachs, et al., Blood, 82:15 (1993)). For instance, they can be triggered by hormonal stimuli, such as glucocorticoid hormones for immature thymocytes, as well as withdrawal of certain growth factors (Watanabe-Fukunaga, et al., Nature, 356:314-317 (1992)). Also, some identified oncogenes such as myc, rel, and E1A, and tumor suppressers, like p53, have been reported to have a role in inducing apoptosis. Certain chemotherapy drugs and some forms of radiation have likewise been observed to have apoptosis-inducing activity (Thompson, Supra). Apoptosis is also triggered by the activation of a family of cysteine proteases having specificity for aspartic acid residues, including Ced-3 of C. elegans, CCP32 (now caspase-3), Yarna/Apopain of humans, and DCP-1 of Drosophila. These proteases are designated as caspases (Alnemri, et al., Cell, 87:171, (1996)).
The Apoptosis-Inducing Signaling Complex
As presently understood, the apoptosis program contains at least three important elements--activators, inhibitors, and effectors. In C. elegans, these elements are encoded respectively by three genes, Ced-4, Ced-9 and Ced-3 (Steller, Science, 267:1445 (1995); Chinnaiyan, et al., Science, 275:1122-1126 (1997)). Two genes, Ced-3 and Ced-4, are required to initiate apoptosis (Yuan and Horvitz, Development 116:309-320, (1990)). Ced-9, which functions upstream of Ced-3 and Ced-4, negatively regulates the apoptotic program by preventing activation of Ced-3 and Ced-4 (Hengartner, et al., Cell 76:665-676 (1994)).
The apoptotic program delineated in C. elegans is conserved in mammalian cells which contain homologues of Ced-9 and Ced-3. One of these homologues, Bcl-2, can partially substitute for Ced-9 in preventing apoptosis in C. elegans (Hengartner and Horvitz, 1994, Cell 76:665-676). The other homologues are cysteine proteases that are closely related to Ced-3, including caspase-3 (Yuan, et al., Cell 75:641-652 (1993): Xue, et al., Genes & Dev. 10:1073-1083 (1996)). Ced-4 is the only remaining C. elegans general apoptosis gene of which the mammalian counterpart had not been found. This gene is believed to function downstream of Ced-9 but upstream of Ced-3 in the C. elegans apoptosis pathway (Shaham and Horvitz, Genes & Dev. 10:578-591, (1996), Cell 86:201-208 (1996)).
In mammalian cells, caspase-3 normally exists in the cytosolic fraction as a 32 kDa inactive precursor which is converted proteolytically to a 20 kDa and a 10 kDa active heterodimer when cells are signaled to die (Schlegel, et al., Biol. Chem. 271:1841-1844, (1996); Wang, et al., EMBO J. 15:1012-1020, (1996)). Bcl-2, located on the outer membrane of mitochondria, prevents the activation of caspase-3. It appears to do this by blocking the mitochondria from releasing cytochrome c, a necessary co-factor for caspase-3 activation (Liu, et al., Cell 86:147-157, (1996); Yang, et al., Science 275:1129-1132, (1997); Kluck, et al., Science 275:1132-1136, (1997)). Deletion of caspase-3 from the mouse genome through homologous recombination results in excessive accumulation of neuronal cells, due to a lack of apoptosis in the brain (Kuida, et al., Nature 384:368-372 (1996). Addition of active caspase-3 to normal cytosol activates the apoptotic program (Enari, et al., Nature 380:723-726 (1996)). Thus, caspase-3 is both necessary and sufficient to trigger apoptosis.
Identified substrates for caspase-3 include poly (ADP-ribose) polymerase (PARP), sterol-regulatory element binding proteins (SREBPs), the U1-associated 70 kDa protein, N4-GD1, huntingtin and DNA dependent protein kinase (Casicola-Rosen, et al., J. Exp. Med. 183:1957-1964(1996); Na, et al., J. Biol. Chem. 271:11209-11213 (1996); Goldberg, et al., Nat. Genet. 13(4):442-449 (1996); Wang, et al. EMBO J. 15:1012-1020 (1996); Nicholson, et al., Nature 376:37-43, (1995)).
Applicants recently established an in vitro apoptosis system to study apoptosis using cytosolic fractions from normally growing HeLa cells. Using this system, two protein factors involved in mammalian apoptosis were identified: cytochrome c (Liu, et al., 1996, Supra) and DNA fragmentation factor (DFF), a novel heterodimer of 45 kDa and 40 kDa subunits. DFF functions downstream of caspase-3 to trigger fragmentation of genomic DNA into nucleosomal segments, a hallmark of apoptosis (Wyllie, Nature 284:555-556, (1980); Liu, et al., 1997 Supra).